1. Technical Field
This invention relates to a method to assess oxidative stress in vivo by quantification of prostaglandin-like compounds and their metabolites produced by a noncyclooxygenase free radical catalyzed mechanism.
2. Prior Art
Free radicals derived primarily from oxygen have been implicated in the pathophysiology of a number of human diseases, such as atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, cancer and aging. A variety of methods have been developed to assess oxidative stress; however, some of these methods have limited sensitivity or specificity, while others are either too invasive or not adaptable for human investigation Halliwell et al., 1987!.
Free radicals are generally short lived and thus, indirect methods of detection are required Pryor, 1989!. Standard detection methods include: electron spin resonance (directly), electron spin resonance (spin trapping), thiobarbituric acid reactive substances (TBARS), detection of malonaldehyde by direct methods (such as HPLC of malonaldehyde itself or as its dinitrophenylhydrazone derivative), detection of other oxidation products from polyunsaturated fatty acids (such as 4-hydroxynonenal), measurement of lipid hydroperoxides, detection of volatile hydrocarbons (ethane, pentane and ethylene), detection of oxidation products from lipids other than polyunsaturated fatty acids (e.g., cholesterol), oxidation of methional, methionine, or 2-keto-4-thiomethylbutanoic acid to ethylene, oxidation of benzoic acid to carbon dioxide (often with radiolabeled carbon dioxide), oxidation of phenol, benzoic acid, or aspirin to hydroxylated products, determination of decreases in antioxidant levels (e.g., decreased GSH, tocopherol, or ascorbate) or of increases in the oxidized products from antioxidants (e.g., tocopherol quinone or the ascorbyl radical), detection of oxidized DNA bases (e.g., thymine glycol, 8-hydroxydeoxyguanosine), detection of oxidized products from proteins (e.g., methionine sulfoxide from methionine) or of proteins oxidized to carbonyl-containing products that then react with hydride-reducing agents, detection of adducts of DNA bases (e.g., by enzymatic hydrolysis post-labeling using P32), and chemiluminescence methods. Id.
Unfortunately, oxidative stress is difficult to assess in humans due to lack of reliable methods to assess oxidant stress in vivo. As one author stated, "one of the greatest needs in the field now is the availability of a non-invasive test to probe the oxidative stress status of humans."Id.
Morrow et al. (1990b) discovered that a series of (F.sub.2 -IPs) prostaglandin F.sub.2 -like compounds, now termed F.sub.2 -isoprostanes, were generated in human plasma during storage at -20.degree. C. for several months or in plasma that had been repeatedly frozen and thawed. Morrow et al. (1990b) determined that these compounds were formed by a non-cyclooxygenase mechanism by autoxidation of arachidonic acid contained in plasma. This article demonstrated that prostaglandin-like compounds could be generated by autoxidation during storage of biological samples which could result in artifactual results with measurements of prostaglandins in stored samples. At that time, there was nothing to suggest this was anything more than just a non-enzymatic in vitro artifact or phenomenon that occurred during the storage of plasma or other lipid containing biological fluids. In fact, this process, autoxidation of lipids or fats, is a major process responsible for spoilage of food during storage.
Formation of F.sub.2 -isoprostanes occurs independently of the cyclooxygenase enzyme and proceeds through intermediates comprising peroxyl radical isomers of arachidonic acid, which undergo endocyclization to form bicyclic endoperoxides. The endoperoxides are then reduced to F.sub.2 -IPs. The endoperoxides also undergo rearrangement in vivo to form D- and E-ring IPs Morrow, 1994a!. Four positional isomers of IPs are formed, each of which can comprise eight racemic diastereomers. IPs are initially formed esterified to phospholipids and subsequently released pre-formed Morrow, 1992a!. Based on the mechanism of formation of IPs, i.e., the formation of compounds with the side chains oriented cis in relation to the cyclopentane ring are highly favored Morrow et al. 1990b!, one compound that would be expected to be formed would be 8-iso-PGF.sub.2.alpha.. Applicants demonstrated that 8-iso-PGF.sub.2.alpha. is, in fact, one of the more abundant F.sub.2 -IPs produced in vivo Morrow et al., 1994b!. There has been considerable interest in this molecule because it exerts biological activity, e.g., it is a potent vasoconstrictor in the lung and kidney Takashashi, et al. 1992; Banerjee et al., 1992!. Furthermore, it has been suggested that the biological effects of 8-iso-PGF.sub.2.alpha., may result from an interaction with a unique receptor Fukunaga et al., 1993!.
It has been recognized that one of the greatest impediments in the field of free radical research has been the lack of reliable methods to assess oxidant stress status in humans Gutteridge et al., 1990!. A considerable body of evidence has accumulated indicating that measurement of F.sub.2 -IPs provide a valuable and reliable approach to assess oxidant stress in vivo both in animal models of oxidant injury and in humans Morrow et al., 1992b; Morrow et al.; 1995!. In this regard, however, quantification of unmetabolized IPs has certain limitations. First, F.sub.2 -IPs can be artifactually generated ex vivo, e.g. in plasma, by auto-oxidation of plasma arachidonic acid if appropriate precautions are not taken Morrow et al., 1990b!. In addition, quantification of F.sub.2 -IPs esterified in tissues or circulating in plasma only provides information at a single point in time rather than an integrated index of IP production. Having a means to obtain an integrated index of oxidant stress status would be very valuable in situations in which the level of oxidant stress fluctuates over time. In this regard, analogous to quantification of urinary metabolites of cyclooxygenase-derived prostanoids Roberts, 1987!, the measurement of the urinary excretion of F.sub.2 -IPs provide a reliable and integrated index of oxidative stress status in vivo.
Applicants have previously identified urinary metabolites of F.sub.2 -IPs that copurify through a mass spectrometric assay developed for quantification of the major urinary metabolite of cyclooxygenase-derived PGD.sub.2 Awad et al., 1993; Morrow, 1991!. However, applicants did not know the parent compounds from which these F.sub.2 -IP metabolites derive. Furthermore, applicants have found that a metabolite of cyclooxygenase-derived PGF.sub.2.alpha., 9.alpha., 11.alpha.-dihydroxy-15-oxo-13,14-dihydro-2,3,18,19-tetranorprost-1,20-dioi c acid, co-chromatographs on capillary gas chromatograph (GC) with these F.sub.2 -IP metabolites. This latter finding confounds an interpretation as to whether an increase in the intensity of these peaks when analyzed by GC and mass spectrometry (MS) represents overproduction of F.sub.2 -IPs or PGF.sub.2.alpha.. Thus, 8-iso-PGF.sub.2.alpha., was studied in order to identify its major metabolites found in human urine as a basis for the development of methods of assay for its quantification to assess oxidative stress in humans.